The present invention relates to purified hydroxylase and flavoprotein components of the enzyme which may be found, for example, in the soluble fraction of Methylobacterium organophilum (CRL.26) (NNRL B-11,222). This invention also relates to a process for oxidizing organic substrates using a mixture of these two components.
Methane is one of the most inexpensive carbon sources for microbial growth. It is known that there are many microorganisms capable of growing on a culture medium in the presence of methane as the principal carbon source. However, not all of these microorganisms share good growth characteristics. It is also known that methane-grown microorganisms can be used to convert methane to methanol under aerobic conditions.
These methane-utilizing microorganisms are generally known as "methylotrophs". The classification system for methylotrophs proposed by R. Whittenbury et al. (J. Gen. Microbiology, 61, 205-218 (1970)) is the most widely recognized. In their system, the morphological characteristics of methane-oxidizing bacteria are divided into five groups: Methylosinus, Methylocystis, Methylomonas, Methylobacter and Methylococcus.
Recently, Patt et al. (International J. Systematic Bacteriology, 26, 226-229 (1976) disclosed that methylotrophic bacteria are those bacteria that can grow non-autotrophically using carbon compounds containing one or more carbon atoms but containing no carbon-carbon bonds. Patt et al. have proposed that methylotrophs should be considered "obligate" if they are capable of utilizing only carbon compounds containing no carbon-carbon bonds (e.g., methane, methanol, dimethylether, methylamines, etc.) as the sole sources of carbon and energy, whereas "facultative" methylotrophs are those organisms that can use both compounds containing no carbon-carbon bonds as well as compounds having carbon-carbon bonds as the sources of carbon and energy. In their paper, Patt et al. disclosed a methane-oxidizing bacterium, which they identified as Methylobacterium organophilum sp nov. (ATCC 27,886). This bacterium presumably differs from all previously described genera and species of methane-oxidizing bacteria because of its ability to utilize a variety of organic substrates with carbon-carbon bonds as sources of carbon and energy.
On the basis of .sup.18 O.sub.2 incorporation from .sup.18 O.sub.2 into the cellular constituents of Pseudomonas methanica, Leadbetter et al. (Nature, 184, 1428-1429 (1959)) suggested that the initial oxidative attack on methane involves an oxygenase. Higgins et al. (J. Biochem., 118, 201-208 (1970) isolated CH.sub.3.sup.18 OH as the product of methane oxidation when suspensions of Pseudomonas methanica or Methanomonas methanooxidans were allowed to oxidize methane in .sup.18 O.sub.2 -enriched atmospheres. The subsequent observation of methane-stimulated NADH oxidation catalyzed by extracts of Methylococcus capsulatus by Ribbons (J. Bacteriol., 122, 1351-1363 (1975)) suggested that the enzyme responsible for this oxygenation is a monooxygenase. These workers relied on indirect enzyme assays, measuring methane-stimulated NADH disappearance spectrophotometrically or methane-stimulated O.sub.2 disappearance polarographically. Recently, methane monooxygenase systems were partially purified from the particulate fraction of Methylosinus trichosporium OB3b by Tonge et al., (J. Biochem., 161, 333-444 (1977) and FEBS Lett., 58 293-299 (1975)). Tonge et al. identified three components required for enzyme activity.
Hutchinson et al. (J. Theor. Biol., 58, 325-335 (1976)) and Colby et al. (J. Biochem., 157, 495-497 (1976)) reported that ethylene is oxidized by the soluble methane monooxygenase from Methylococcus capsulatus Strain Bath. The latter investigators reported that the "particulate membrane preparations" of Methylococcus capsulatus Strain Bath did not have methaneoxygenase activity as determined by the bromomethane disappearance test.
Subsequently, Colby et al. (J. Biochem., 165, 395-402 (1977)) disclosed that the soluble fraction of the obligate Methylococcus capsulatus Strain Bath is a very non-specific oxygenase in that it oxidizes alkanes to alcohols, alkenes to 1,2-epoxides, dimethylether to ethanol and ethanal, styrene to styrene epoxide, and pyridine to pyridine N-oxide.
Most recently, Stirling et al. (J. Biochem., 96, 205 (1979) and J. Gen. Microbiol., 116, 277 (1980)) reported that the obligate methane-utilizing methylotroph, Methylosinus trichosporium OB3b, contained a soluble methane monooxygenase activity similar to that of the soluble methane monooxygenase from the Methylococcus capsulatus Strain Bath. U.K. Pat. No. 1,603,864 discloses a process for oxidation of selected organic substrates employing Methylococcus capsulatus or Methylosinus trichosporium as soluble fractions.
The methane monooxygenase enzyme from the organism Methylococcus capsulatus (Bath Strain) has been resolved into three component by DEAE-cellulose chromatography by Colby et al. (Biochem. J., 171, 461 (1978)), Dalton (Advances in Applied Microbiology, 26, 71 (1980)) and Dalton et al. (Flavins and Flavoproteins, Massey and Williams, eds., Chapter 128, p. 763.) In the latter article the authors indicate that all three components A (a hydroxylase), B (an enzyme of molecular weight of about 15,000) and C (a flavoprotein) are necessary to catalyze the reductive oxygenation of the hydrocarobn substrate. The component A derived from the Bath Strain contains two subunits and has a molecular weight of about 200,000.